A method for detecting chlorpyrifos in tea leaves

Abstract

The application discloses a method for detecting chlorpyrifos in tea, and adopts a microwave method to quickly prepare gold-doped carbon dots (CD Au ) with fullerene as a precursor, uses Victoria blue B (VBB) as a probe molecule, and constructs a simple, sensitive, quick and complex pretreatment-free SERS method for quickly detecting chlorpyrifos in tea by combining surface-enhanced Raman scattering (SERS) with aptamer-regulated gold-doped carbon dot catalytic reaction.

Description

[Technical Field]

[0002] This invention relates to a method for detecting chlorpyrifos poisoning in tea by combining a SERS substrate with carbon dot catalysis, belonging to the field of Raman spectroscopy analysis and detection technology. [Background Technology]

[0004] Chlorpyrifos is a moderately toxic organophosphate insecticide that is effective against various chewing and sucking pests on crops such as rice, wheat, and tea, as well as tea pests such as the tea geometrid moth and the tea mirid bug. It is widely used for controlling crop pests and household pests. However, chlorpyrifos is persistent in the environment and bioaccumulates, exhibiting genotoxicity such as DNA damage and gene mutation. If chlorpyrifos residues enter the bodies of humans and other mammals through environmental routes, causing poisoning, it may increase the potential risk of cancers such as lung cancer and leukemia, and even lead to neurological dysfunction, paralysis, or death. Long-term low-dose exposure poses a significant threat to human health.

[0005] Chlorpyrifos is widely used for pest and disease control in tea plants, making it prone to residues in tea leaves. Therefore, developing a rapid and simple detection method for chlorpyrifos in tea is of great significance. To prevent potential harm from chlorpyrifos pesticide residues, many methods have been used to detect chlorpyrifos residues in tea. Currently, common methods for detecting chlorpyrifos in tea have the following drawbacks:

[0006] 1. Liquid chromatography cannot directly provide qualitative results for unknown substances from chromatograms; it requires the use of known standards for comparison. When no pure substance is available for reference, qualitative identification becomes very difficult, necessitating the use of mass spectrometry, infrared spectroscopy, and chemical methods in conjunction. Furthermore, most metal salts and substances with poor thermal stability cannot be analyzed, resulting in low sensitivity and limiting its widespread application in trace analysis.

[0007] 2. Gas chromatography-mass spectrometry is not suitable for widespread and flexible use due to the large size of the instruments and the requirement for professional operation; it cannot be used for on-site testing.

[0008] 3. Liquid chromatography-mass spectrometry is a common technique in the field of detection and analysis, but the instrument structure of liquid chromatography-mass spectrometry is relatively complex, the price is relatively expensive, the operation is complicated, the detection speed is slow, it is time-consuming, and it requires professional personnel to operate, so it is not suitable for widespread and flexible use.

[0009] 4. The application of SERS technology to chlorpyrifos poisoning in tea requires the combination of many complex density functional theories or other complex computational theoretical models, which makes the method not simple enough and the detection efficiency not high.

[0010] 5. Currently, after tea samples are prepared for testing, they cannot be used for subsequent chlorpyrifos tests after the first test, resulting in a low sample recovery rate.

[0011] Currently, the use of aptamers as a molecular recognition method is a research hotspot. Their specificity and high affinity for binding to targets are comparable to or even superior to antibodies. Furthermore, they offer advantages such as simple and rapid synthesis, wide applicability, ease of chemical modification, and multifunctionality. Combining aptamers with SERS technology for pesticide residue detection holds great promise. This application will utilize aptamer-regulated SERS substrate and carbon dot catalysis to establish a simple, rapid, highly sensitive, and selective SERS method for detecting chlorpyrifos in tea, requiring no complex pretreatment. [Summary of the Invention]

[0013] To address the shortcomings of existing technologies, the present invention aims to provide a simple, sensitive, convenient, and non-complex pretreatment method for detecting chlorpyrifos poisoning in tea by combining aptamer-regulated SERS substrate with carbon dot catalysis.

[0014] The technical solution to achieve the objective of this scheme is: a method for detecting chlorpyrifos poisoning in tea, comprising fullerene (C... 60 Configuration, gold-doped carbon dots (CD) Au The preparation of chlorpyrifos and its determination by SERS are as follows:

[0015] (1) Take a certain mass of fullerene (C 60 Dissolve a suitable volume of toluene in a solution, add a certain volume of ultrapure water, and sonicate in an ultrasonic cleaner until all the toluene evaporates, resulting in a deep yellow suspension, which is the fullerene solution. Because fullerenes are lipophilic nanomaterials with poor solubility in most polar solvents, toluene is chosen to promote complete dissolution of fullerenes, improve their dispersibility, and enhance subsequent detection resolution; furthermore, fullerene (C... 60 Carbon is a non-metallic element, a molecule composed of 60 carbon atoms. It is a stable molecule formed by the combination of carbon atoms. It has 60 vertices and 32 faces, mostly stable pentagons and hexagons. These properties improve its activity as a heterogeneous carbon catalyst, making it a highly efficient catalyst.

[0016] (2) Take an appropriate amount of the above fullerene (C 60 The solution was prepared by adding HAuCl4 solution and then ultrasonically dissolving it in deionized water in an ultrasonic cleaner to obtain a transparent solution.

[0017] (3) Transfer the above transparent solution into a digestion vessel, seal it, and place it in a microwave digestion apparatus. The microwave digestion heating time is 15 minutes. After the reaction is complete, remove it and cool it to room temperature to obtain a light red clear solution, denoted as CD. Au ;

[0018] (4) Add 0–0.30 mmol / L of chlorpyrifos aptamer solution and 0–34 μg / mL of CD to a 100 mL Erlenmeyer flask. Au Mix the solution thoroughly and allow it to stand to react;

[0019] (5) After the reaction of the solution in step (4) is completed, add an appropriate concentration of chlorpyrifos, mix and let stand for 9 minutes to allow the aptamer to fully bind with the target substance, thereby allowing the aptamer to be released from CD. Au Surface detachment, CD Au The catalytic activity will increase, and the SERS intensity of the system will rise;

[0020] (6) Then add 0–60 mmol / L glucose as a reducing agent, 0–1.18 mmol / L HCl, and 0–0.90 g / L HAuCl4 solution, mix well and make up to volume. Glucose is not only inexpensive but also an environmentally friendly reagent. HCl ionizes to release a large amount of Cl. - Cl - In the presence of these elements, gold ions aggregate to form a gold nanoparticle SERS substrate, CD Au It has a strong catalytic effect on the SERS substrate of gold nanoparticles. At the same time, due to the large specific surface area of ​​gold nanoparticles, the contact area is also increased, resulting in a better SERS signal. In the reaction system of glucose reducing chloroauric acid, the pH value of the system affects the size and morphology of the produced gold nanoparticles. Under alkaline conditions, glucose will undergo tautomerism and rearrangement between aldose and ketose, reducing the number of aldehyde groups that play a reducing role. The addition of HCl can be used to control the pH of the system, thereby controlling the generation of gold nanoparticles.

[0021] (7) Heat the above mixed solution in a water bath at 50-100°C for 0-24 minutes, and terminate the reaction with ice water to quickly lower the temperature and prevent subsequent slow reactions.

[0022] (8) Take an appropriate amount of solution after terminating the reaction in step (7) and add 0 to 0.17 μmol / L Victoria Blue B (VBB) for SERS detection. Victoria Blue B (VBB) is added as the probe molecule of the system, i.e. the indicator of the Raman signal.

[0023] (9) Detection at 1616cm -1 The surface-enhanced Raman scattering peak intensity I at the Raman shift 1616cm-1 In addition, without adding chlorpyrifos solution as a blank, its blank value I0 was measured, and ΔI was calculated.1616cm-1 = I 1616cm-1 - I0 value. During data acquisition, the Raman spectrometer parameters were set as follows: laser wavelength 633 nm, laser power 3.0 mW, and acquisition time 10 s;

[0024] (10) A standard curve was plotted using known chlorpyrifos content, and the chlorpyrifos content in the supernatant of the tea sample was determined using the working curve method. The preparation process of the supernatant of the tea sample includes the following steps:

[0025] S1: Weigh 5 g of tea sample and grind it into powder;

[0026] S2: Place in a 50 mL centrifuge tube, add 25 mL acetone, vortex for 1 min, and sonicate for 2 min;

[0027] S3: Then put it into a centrifuge and centrifuge at 4200 r / min for 5 min. Take 1 mL of the suspension and make up to 20 mL as the sample solution.

[0028] S4: Measure 6 mL of sample solution into a 10 mL centrifuge tube, weigh 0.28 g of iron(III) oxide, 0.13 g of graphitized carbon, and 600 mg of anhydrous magnesium sulfate and add them to the 6 mL sample solution. Mix and shake for 1 min, then centrifuge at 4500 r / min for 5 min to remove interference from chlorophyll, minerals, tea polyphenols, carbohydrates and other substances in the matrix, and prepare the tea supernatant.

[0029] Preferably, in step (1), the ratio of fullerene mass: toluene volume: ultrapure water volume = 0.02 g: 20 ml: 100 mL, and the prepared fullerene solution is 0.2 g / L.

[0030] Preferably, in step (2), the fullerene (C 60 Take 1.5 ml of the solution, the mass concentration of HAuCl4 is 0.01 g / mL, the volume used is 70 μL, and the amount of deionized water used is 20 mL.

[0031] Preferably, in step (3), the microwave digestion process is carried out at a temperature of 180°C and a pressure of 1.0 MPa.

[0032] Preferably, in step (4), the chlorpyrifos aptamer solution is 0.14 mmol / L, and CD Au The solution was mixed with 18 μg / L and allowed to stand for 5 minutes to allow the aptamer to be more completely and better adsorbed onto the surface of the carbon dots.

[0033] Preferably, in step (6), the glucose concentration is 45 mmol / L, the HCl concentration is 0.59 mmol / L, the HAuCl4 concentration is 0.65 g / L, and the solution is mixed and diluted to 10 mL.

[0034] Preferably, in step (7), the water bath heating temperature is 70°C and the reaction time is 18 min.

[0035] Preferably, in step (8), 2 mL of the solution after the reaction is terminated in step (7) is taken, and the solubility of Victoria Blue B (VBB) solution is 0.125 μmol / L.

[0036] The present invention provides a method for detecting chlorpyrifos poisoning in tea based on SERS substrate regulation combined with carbon dot catalysis, which has the following advantages:

[0037] 1. Under water heating conditions at 70℃, CD Au The reaction of glucose and HAuCl4 to generate gold nanoparticles as a SERS substrate exhibits strong catalytic activity: fullerenes possess photoinduced electron transfer and a large specific surface area, while metal nanoparticles, due to their small size and large specific surface area, possess unique physicochemical properties. Doping fullerenes with gold forms C-Au bonds, and the catalytic activities of both work synergistically, accelerating electron transfer in the chemical reaction and enabling the system to generate gold nanoparticles more quickly.

[0038] 2. This invention utilizes aptamer-regulated SERS substrate and carbon dot catalysis to establish a simple, sensitive, rapid, and complex pretreatment-free method for detecting chlorpyrifos poisoning in tea leaves using SERS.

[0039] 3. The recovery rates of black tea, white tea and green tea were verified to be 96.80-102.13%, with relative standard deviations of 2.33-4.04%. The tea samples showed high recovery rates and strong resistance to interference. [Attached Image Description]

[0041] Figure 1 Schematic diagram of the principle for SERS determination of trace chlorpyrifos;

[0042] Figure 2 To disable CD for chlorpyrifos aptamers Au SERS spectrum of the catalytic reaction system (a: 0 ng / L chlorpyrifos + 0 mmol / L chlorpyrifos aptamer + 0.018 mg / L LCD) Au+ 0.65 g / L HAuCl4 + 45 mmol / L glucose + 0.59 mmol / L HCl + 0.125 μmol / L VBB; b: 0 ng / L chlorpyrifos + 0.14 mmol / L chlorpyrifos aptamer + 0.018 mg / LCD Au + 0.65 g / L HAuCl4 + 45 mmol / L glucose + 0.59 mmol / L HCl + 0.125 μmol / L VBB);

[0043] Figure 3 To disable CD for chlorpyrifos aptamers Au Absorption spectrum of the catalytic reaction system (a: 0 ng / L chlorpyrifos + 0 mmol / L chlorpyrifos aptamer + 0.018 mg / L LCD) Au + 0.65 g / L HAuCl4 + 45 mmol / L glucose + 0.59 mmol / L HCl + 0.125 μmol / L VBB; b: 0 ng / L chlorpyrifos + 0.14 mmol / L chlorpyrifos aptamer + 0.018 mg / LCD Au + 0.65 g / L HAuCl4 + 45 mmol / L glucose + 0.59 mmol / L HCl + 0.125 μmol / L VBB);

[0044] Figure 4 To disable CD for chlorpyrifos aptamers Au RRS spectrum of the catalytic reaction system (a: 0 ng / L chlorpyrifos + 0 mmol / L chlorpyrifos aptamer + 0.018 mg / L LCD) Au + 0.65 g / L HAuCl4 + 45 mmol / L glucose + 0.59 mmol / L HCl + 0.125 μmol / L VBB; b: 0 ng / L chlorpyrifos + 0.14 mmol / L chlorpyrifos aptamer + 0.018 mg / LCD Au + 0.65 g / L HAuCl4 + 45 mmol / L glucose + 0.59 mmol / L HCl + 0.125 μmol / L VBB);

[0045] Figure 5 Turn on CD for different concentrations of chlorpyrifos Au SERS spectrum of the catalytic reaction system;

[0046] Figure 6 Turn on CD for different concentrations of chlorpyrifos AuUV absorption spectrum of the catalytic reaction system (a: 0 ng / L chlorpyrifos + 0.14 mmol / L chlorpyrifos aptamer + 0.018 mg / L CD) Au +0.65 g / L HAuCl4+ 45 mmol / L L-HCl + 0.59 mmol / L HCl + 0.125 μmol / L VBB; b: a+12.8 ng / L chlorpyrifos; c: a+117.8 ng / L chlorpyrifos; d: a+222.8 ng / L chlorpyrifos; e: a+327.8 ng / L chlorpyrifos; f: a+432.8 ng / L chlorpyrifos).

[0047] Figure 7 Turn on CD for different concentrations of chlorpyrifos Au RRS spectrum of the catalytic reaction system (A: 0 ng / L chlorpyrifos + 0.14 mmol / L chlorpyrifos aptamer + 0.018 mg / L CD) Au + 0.65 g / L HAuCl4 + 45 mmol / L glucose + 0.59 mmol / L HCl + 0.125 μmol / L VBB; b. a + 12.8 ng / L chlorpyrifos; c: a + 117.8 ng / L chlorpyrifos; d: a + 222.8 ng / L chlorpyrifos; e: a + 327.8 ng / L chlorpyrifos; f: a + 432.8 ng / L chlorpyrifos).

[0048] Figure 8 Electron microscopy image of gold nanoparticles at a chlorpyrifos concentration of 0 ng / L (0 ng / L chlorpyrifos + 0.14 mmol / L chlorpyrifos aptamer + 0.018 mg / L CD). Au + 0.65 g / L HAuCl4 + 45 mmol / L glucose + 0.59 mmol / L HCl + 0.125 μmol / L VBB);

[0049] Figure 9 shows an electron micrograph of gold nanoparticles at a concentration of 117.8 ng / L chlorpyrifos (117.8 ng / L chlorpyrifos + 0.14 mmol / L chlorpyrifos aptamer + 0.018 mg / L CD). Au +0.65 g / L HAuCl4 + 45 mmol / L glucose + 0.59 mmol / L HCl + 0.125 μmol / L VBB.

[0050] Figure 10Electron microscopy image of gold nanoparticles without chlorpyrifos and aptamer (0 ng / L chlorpyrifos + 0 mmol / L chlorpyrifos aptamer + 18 μg / L + 0.65 g / L HAuCl4 + 45 mmol / L glucose + 0.59 mol / L HCl + 0.125 μmol / L VBB).

[0051] Figure 11 For CD Au Effect of solution concentration on the detection system of chlorpyrifos (432.8 ng / L chlorpyrifos + 0.14 mmol / L chlorpyrifos aptamer + x μg / LCD) Au +0.65 g / L HAuCl4 + 45 mmol / L glucose + 70℃ + 18 min + 0.59 mol / L HCl + 0.125 μmol / L VBB);

[0052] Figure 12 The effect of glucose concentration on the chlorpyrifos detection system is shown in the figure (432.8 ng / L chlorpyrifos + 0.14 mmol / L chlorpyrifos aptamer + 18 μg / L + 0.65 g / L HAuCl4 + x mmol / L glucose + 70℃ + 18 min + 0.59 mmol / L HCl + 0.125 μmol / L VBB).

[0053] Figure 13 The effect of HAuCl4 concentration on the chlorpyrifos detection system is shown in the figure (432.8 ng / L chlorpyrifos + 0.14 mmol / L chlorpyrifos aptamer + 18 μg / L + x g / L HAuCl4 + 45 mmol / L glucose + 70℃ + 18 min + 0.59 mmol / L HCl + 0.125 μmol / L VBB).

[0054] Figure 14 The effect of chlorpyrifos aptamer concentration on the chlorpyrifos detection system is shown in the figure (432.8 ng / L chlorpyrifos + x mmol / L chlorpyrifos aptamer + 18 μg / L + 0.65 g / L HAuCl4 + 45 mmol / L glucose + 70 ℃ + 18 min + 0.59 mmol / L HCl + 0.125 μmol / L VBB).

[0055] Figure 15The graph shows the effect of HCl concentration on the detection system of chlorpyrifos (432.8 ng / L chlorpyrifos + 0.14 mmol / L chlorpyrifos aptamer + 18 μg / L + 0.65 g / L HAuCl4 + 45 mmol / L glucose + 70℃ + 18 min + x mmol / L HCl + 0.125 μmol / L VBB).

[0056] Figure 16 The graph shows the effect of temperature on the chlorpyrifos detection system (432.8 ng / L chlorpyrifos + 0.14 mmol / L chlorpyrifos aptamer + 18 μg / L + 0.65 g / L HAuCl4 + 45 mmol / L glucose + x℃ + 18 min + 0.59 mmol / L HCl + 0.125 μmol / L VBB).

[0057] Figure 17 The effect of time on the detection system of chlorpyrifos (432.8 ng / L chlorpyrifos + 0.14 mmol / L chlorpyrifos aptamer + 18 μg / L + 0.65 g / L HAuCl4 + 45 mmol / L glucose + 70℃ + x min + 0.59 mmol / L HCl + 0.125 μmol / L VBB);

[0058] Figure 18 The effect of VBB concentration on the chlorpyrifos detection system is shown in the figure (432.8 ng / L chlorpyrifos + 0.14 mmol / L chlorpyrifos aptamer + 18 μg / L + 0.65 g / L HAuCl4 + 45 mmol / L glucose + 70℃ + 18 min + 0.59 mmol / L HCl + x μmol / L VBB).

[0059] Figure 19 Working curve for SERS detection of chlorpyrifos ((0, 12.8, 117.8, 222.8, 327.8, 432.8 ng / L) chlorpyrifos + 0.14 mmol / L chlorpyrifos aptamer + 18 μg / L + 0.65 g / L HAuCl4 + 45 mmol / L glucose + 0.59 mmol / L HCl + 0.125 μmol / L VBB).

Detailed Implementation Methods

[0061] The present invention will be further described in detail below with reference to specific embodiments, which do not constitute any limitation on the present invention. The specific sequence strand of the chlorpyrifos aptamer described in all embodiments of the present invention is 5′-DNACCTGCCACGCTCCGCAAGCTTAGGGTTACGCCTGCAGCGATTCTTGATCGCGCTGCTGGTAATCCTTCTTTAAGCTTGGCACCCGCATCGT-3′.

[0062] Example 1

[0063] I. Basic Preparation Method

[0064] (1) Preparation of fullerenes (C 60 )

[0065] Weigh 0.02 g of fullerene and dissolve it in 20 mL of toluene. The solution is bright purple. Add 100 mL of ultrapure water and place the solution in an ultrasonic cleaner and sonicate until all the toluene evaporates. The resulting solution is a dark yellow suspension, yielding a 0.2 g / L fullerene solution.

[0066] (2) CD Au Preparation

[0067] Take the above C 60 1.5 mL of solution was added, followed by 70 μL of 0.01 g / mL HAuCl4 solution. The solution was then ultrasonically dissolved in 20 mL of deionized water in an ultrasonic cleaner to obtain a clear solution. This solution was then transferred to a digestion vessel, sealed, and placed in a microwave digester. The temperature was set to 180 ℃, the pressure to 1.0 MPa, and the microwave digestion time to 15 min. After the reaction was complete, the solution was removed and cooled to room temperature, yielding a pale red, clear solution, denoted as CD. Au The resulting carbon dots are in C 60 The concentration was 13.91 mg / L, which was diluted to 0.6 μg / mL for later use.

[0068] (3) SERS method for determining chlorpyrifos

[0069] Add 140 μL of 0.01 mmol / L chlorpyrifos aptamer solution and 300 μL of 0.6 μg / mL CD to a 100 mL Erlenmeyer flask. AuMix the solution thoroughly and let it stand for 5 minutes. After the reaction is complete, add an appropriate concentration of chlorpyrifos and let it stand for 9 minutes. Then add 900 μL of 0.5 mol / L glucose, 590 μL of 0.01 mol / L HCl, and 650 μL of 0.001 g / mL HAuCl4 solution, mix well, and bring the volume to 10 mL. Incubate in a 70 °C water bath for 18 minutes, and terminate the reaction with ice water. Take 2 mL of the reaction solution and add 25 μL of 1x10... -3 SERS was performed using mol / LVBB. Measurements were taken at 1616 cm⁻¹. -1 The surface-enhanced Raman scattering peak intensity I at the Raman shift 1616cm-1 In addition, without adding chlorpyrifos solution as a blank, its blank value I0 was measured, and ΔI was calculated. 1616cm-1 = I 1616cm-1 - I0 value. During data acquisition, the Raman spectrometer parameters were set as follows: laser wavelength 633 nm, laser power 3.0 mW, and acquisition time 10 s.

[0070] II. Method Principles

[0071] Under water conditions at 70℃, CD Au It exhibits strong catalytic activity in the reaction of glucose and HAuCl4 to generate gold nanoparticle SERS substrates. Under suitable reaction conditions, CD... Au It exhibits strong catalytic activity, but when chlorpyrifos nucleic acid aptamers are added to the system, the aptamers adsorb onto CD. Au The surface of the carbon dots is affected, leading to inhibited catalytic activity and reduced SERS substrate formation in gold nanoparticles, thus weakening the SERS intensity of the system. When the target molecule chlorpyrifos is added to the system, it specifically binds to the aptamer, forming a chlorpyrifos-aptamer complex, which causes the chlorpyrifos nucleic acid aptamer to detach from the CD. Au Surface, CD Au The catalytic effect is restored, and the SERS intensity of the system also increases accordingly. With increasing chlorpyrifos concentration, CD... Au The catalytic effect is relatively enhanced, resulting in more gold nanoparticles and an enhanced SERS signal in the system. The principle is as follows: Figure 1 As shown.

[0072] III. Measurement Results and Analysis

[0073] (1) Chlorpyrifos aptamer shuts down CD Au SERS, RRS, and UV absorption spectra of the catalytic reaction system

[0074] Due to CD Au It exhibits good adsorption properties; chlorpyrifos aptamer is added to the detection system, CD AuThe surface is quickly adsorbed and encapsulated by the aptamer, greatly reducing the contact area with the glucose-HAuCl4 catalytic reaction, inhibiting its catalytic activity, reducing the number of gold nanoparticles generated, and decreasing the SERS strength of the system. Figure 2 .Depend on Figure 2 As shown, it is at 1616cm -1 The surface-enhanced Raman scattering peak at the Raman shift has a relatively large intensity.

[0075] Similarly, the UV absorption spectrum of the system shows a significant decrease in absorption peaks around 305 nm and 568 nm after the addition of the aptamer, as shown in the following results. Figure 3 .

[0076] Similarly, the absorption peaks of the RRS of the system at around 282 nm, 370 nm and 564 nm were significantly reduced after the addition of the aptamer, as shown in Figure 4.

[0077] (2) Different concentrations of chlorpyrifos activated CD Au Catalytic reaction

[0078] Different concentrations (0 ng / L, 12.8 ng / L, 117.8 ng / L, 222.8 ng / L, 327.8 ng / L, 432.8 ng / L) of chlorpyrifos were added to the system. Chlorpyrifos specifically bound to the aptamer, and CD... Au As the system was gradually exposed, its catalytic activity recovered, and the number of gold nanoparticles generated increased. Using VBB as a probe molecule, the SERS intensity of the system was enhanced, as shown in the following results. Figure 5 As shown, by Figure 5 It can be seen that within the concentration range of 12.8–432.8 ng / L, the SERS intensity of chlorpyrifos increases with increasing concentration, showing a positive correlation.

[0079] The ultraviolet absorption spectra of the detection system containing different concentrations of chlorpyrifos were examined, and the results are as follows: Figure 6 As shown, from Figure 6 It can be seen that due to the increase in gold nanoparticles, the ultraviolet absorption signal of the system is enhanced at around 305nm and 568nm.

[0080] The RRS spectra of detection systems with different concentrations of chlorpyrifos were analyzed, and the results are as follows: Figure 7 As shown, by Figure 7 It can be seen that the RRS signal is enhanced at approximately 282nm, 370nm, and 564nm, which is consistent with... Figure 5 The phenomenon of enhanced SERS signal is consistent with that in the middle.

[0081] (3) Scanning electron microscopy test

[0082] In the chlorpyrifos detection platform system, when the concentration of chlorpyrifos in the solution is 0 ng / L, its electron micrograph is as follows. Figure 8 As shown, by Figure 8 It can be concluded that: chlorpyrifos does not bind to the aptamer to release naked CD40. Au At this time, the aptamer is compatible with CD. Au The inhibitory effect is the strongest, and the number of gold nanoparticles generated is the smallest;

[0083] When the concentration of chlorpyrifos in the system was 117.8 ng / L, its electron micrograph was as follows. Figure 9 As shown, by Figure 9 It can be seen that: chlorpyrifos binds to its aptamer, exposing CD4+ cells in the system. Au With the addition of pesticides, the catalytic performance of the system was restored to some extent, and the number of gold nanoparticles generated increased compared to the system without pesticides. When neither aptamers nor pesticides were present in the system, its electron micrograph is shown below. Figure 10 As shown, by Figure 10 It can be seen that the system generated a large number of gold nanoparticles, indicating that CD Au It exhibits good catalytic performance in the system and is a good catalyst. Results show that chlorpyrifos can bind to the aptamer, thereby inhibiting CD. Au Its catalytic properties can indirectly regulate the number of gold nanoparticles generated.

[0084] (4) Optimization of factors affecting the system

[0085] For CD respectively Au The concentrations of the solutions, glucose solution, HAuCl4 solution, chlorpyrifos aptamer solution, and HCl solution were optimized. The effects of different glucose concentrations on the chlorpyrifos detection system are shown in the following results. Figure 11 The effects of different glucose concentrations on the chlorpyrifos detection system are as follows: Figure 12 The effects of different HAuCl4 concentrations on the chlorpyrifos detection system are as follows: Figure 13 The effects of different chlorpyrifos aptamer concentrations on the chlorpyrifos detection system are as follows: Figure 14 The effects of different HCl concentrations on the chlorpyrifos detection system are as follows: Figure 15 ,Depend on Figure 11-15 It can be known that CD Au When the concentrations of the solutions are 18 μg / L, 45 mmol / L, 0.65 g / L, 0.14 mmol / L, and 0.59 mmol / L, respectively, the ΔI of the system is... 1616cm-1 The maximum value was reached. Therefore, the above conditions were selected as the optimal detection conditions in the final experiment. After optimizing the detection system with the above conditions, the time, water temperature, and humidity were further optimized. The effects of different temperatures on the chlorpyrifos detection system are shown in the following figures. Figure 16As shown, the effects of different time points on the chlorpyrifos detection system are as follows: Figure 17 As shown, from Figure 16-17 It can be seen that when the water bath temperature is 70℃ and the reaction time is 18 min, the system's ΔI 1616cm-1 To reach the maximum value, a reaction temperature of 70℃ and a reaction time of 18 min were selected. Finally, the VBB probe molecule was optimized, and the test results are as follows: Figure 18 As shown, by Figure 18 It can be seen that when the VBB concentration in the detection system is 0.125 μmol / L, the system ΔI 1616cm-1 Since the maximum value was reached, the VBB concentration in the system was selected to be 0.125 μmol / L.

[0086] (5) Working curve

[0087] According to the experimental method, chlorpyrifos (ng / L) was used to control ΔI. 1616cm -1 Plot the working curve, such as Figure 19 As shown. Experimental results indicate that chlorpyrifos in the range of 12.8–432.8 ng / L is related to ΔI... 1616cm -1 The relationship is linear, and its regression linear equation is ΔI. 1614cm -1 =4.7812X - 0.2417, correlation coefficient R 2 = 0.9961, detection limit is 2.40 x 10⁻⁶ -7 mg / kg.

[0088] (6) Interference experiment

[0089] The common ion (Mg) was investigated according to the experimental method. 2+ Na + Cu 2+ Zn 2+ Ca 2+ Ba 2+ K + NH4 + CO3 2- SO4 2- Cl - HCO 3- The interference of co-application of pesticides (bifenthrin, imidacloprid, pyridaben, and acetamiprid) on the detection system at a chlorpyrifos concentration of 222.8 ng / mL was investigated. The chlorpyrifos concentration was 222.8 ng / L, and common ions, including 22.28 μg / L of Mg, were added simultaneously with the chlorpyrifos. 2+ Na + Cu 2+ Zn 2+ Ca 2+Ba 2+ K + NH4 + CO3 2- SO4 2- Cl - HCO 3- Or co-application of pesticides: 22.28 μg / L of bifenthrin, imidacloprid, pyridaben, and acetamiprid. The interference of common ions and co-application of pesticides on the detection system was investigated. The effects of interfering substances are shown in Table 1. From the experimental results in Table 1, it can be seen that when 22.28 μg / L of Mg... 2+ Na + Cu 2+ Zn 2 + Ca 2+ Ba 2+ K + NH4 + CO3 2- SO4 2- Cl - HCO 3- The assay did not interfere with the determination; 22.28 μg / L bifenthrin, imidacloprid, pyridaben, and acetamiprid did not interfere with the determination; thus, this method has good selectivity.

[0090] Table 1 Effects of Interfering Substances

[0091]

[0092] (7) Analysis of dried tea leaves of black tea, white tea and green tea

[0093] Weigh 5 g each of dried black tea, white tea, and green tea, grind them into powder, place them in a 50 mL centrifuge tube, add 25 mL of acetone, vortex for 1 min, ultrasonically extract for 2 min, then centrifuge at 4200 r / min for 5 min. Take 1 mL of the suspension and dilute to 20 mL to prepare a 222.8 ng / L chlorpyrifos standard solution. Measure 6 mL of the 222.8 ng / L chlorpyrifos standard solution into a 10 mL centrifuge tube, weigh 0.28 g of iron(III) oxide, 0.13 g of graphitized carbon, and 600 mg of anhydrous magnesium sulfate, add them to the 6 mL of standard solution, mix and vortex for 1 min, centrifuge at 4500 r / min for 5 min to remove interference from chlorophyll, minerals, tea polyphenols, carbohydrates, and other substances in the matrix. Take the supernatant and perform a spiked recovery experiment on the green tea sample in 5 parallel runs according to the SERS spectral acquisition experimental method of the chlorpyrifos detection platform. As shown in Table 2, five parallel spiked recovery experiments were conducted on dry green tea leaves according to the experimental method. The recovery rate reached 96.80%–102.13%, and the standard deviation of the recovery was 2.33%–4.04%.

[0094] Table 2 Sample Analysis

[0095] Table 3-2 Sample analysis

[0096]

[0097] The above description is a detailed explanation of the preferred embodiments of the present invention. However, the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications made under the technical spirit of the present invention should fall within the patent scope covered by the present invention.

Claims

1. A method for detecting chlorpyrifos poisoning in tea, comprising the following steps: including fullerene preparation, gold-doped carbon dot CD... Au The preparation and SERS determination of chlorpyrifos are carried out using the following specific steps: (1) take a certain amount of fullerene dissolved in an appropriate volume of toluene, add a certain volume of ultrapure water, placed in ultrasonic cleaning instrument ultrasonic toluene volatilization, get solution is dark yellow suspension, get fullerene solution; (2) take appropriate amount of the above fullerene solution, then add HAuCl4 solution, placed in ultrasonic cleaning instrument ultrasonic dissolution in deionized water, get transparent solution; (3) The above transparent solution was transferred into a digestion tank, sealed and placed in a microwave digestion instrument, and the microwave digestion heating operation time was 15 min. After waiting for the reaction to end, it was taken out and cooled to room temperature to obtain a light red clear solution, which was recorded as CD Au ; (4) In 100 mL conical flask, add 0-0.30 mmol / L concentration of chlorpyrifos aptamer solution and 0-34 μg / mL concentration of CD Au Mix the solution and let it stand for reaction; (5) after the completion of step (4) solution reaction, add appropriate amount of chlorpyrifos mixed and placed for 9 min; (6) then add 0-60 mmol / L of glucose as reducing agent and 0-1.18 mmol / L of HCl, 0-0.90 g / L of HAuCl4 solution, mix well and constant volume; (7) the above mixed solution is heated in water bath for 50-100 DEG C for 0-24 min, the reaction is terminated by ice water, the temperature is quickly reduced to prevent subsequent slow reaction; (8) take appropriate amount of step (7) solution after termination of the reaction, add 0-0.17 μmol / L of victoria blue B for SERS detection, add victoria blue B as the probe molecule of the system; (9) Detection at 1616cm -1 The surface-enhanced Raman scattering peak intensity I at the Raman shift 1616cm-1 In addition, without adding chlorpyrifos solution as a blank, its blank value I0 was measured, and ΔI was calculated. 1616cm-1 = I 1616cm-1 - I0 value, during data acquisition, the parameters of the Raman spectrometer were set as follows: laser wavelength 633 nm, laser power 3.0 mW, acquisition time 10 s; (10) a known content of chlorpyrifos is used to draw a standard curve, and the content of chlorpyrifos in the supernatant of tea sample is determined by the working curve method, and the preparation process of the supernatant of tea sample comprises the following steps: S1: weigh 5 g of tea sample and grind into powder; S2: put into 50 mL centrifuge tube, add 25 mL of acetone, whirlpool for 1 min, and ultrasonic extraction for 2 min; S3: then put into the centrifuge at 4200 r / min for 5 min, take 1 mL of the suspension and constant volume to 20 mL as the sample solution; S4: take 6 mL of sample solution in a 10 mL centrifuge tube, weigh 0.28 g of ferric oxide, 0.13 g of graphitized carbon and 600 mg of anhydrous magnesium sulfate, add to 6 mL of sample solution, mix and shake for 1 min, centrifuge at 4500 r / min for 5 min, remove the interference of chlorophyll, minerals, tea polyphenols and carbohydrates in the matrix to prepare the tea supernatant.

2. The method for detecting chlorpyrifos in tea leaves according to claim 1, characterized in that: In step (1), the mass of fullerene: volume of toluene: volume of ultrapure water = 0.02 g: 20 ml: 100 mL, and the prepared fullerene solution is 0.2 g / L.

3. The method for detecting chlorpyrifos in tea leaves according to claim 1, characterized in that: In step (2), the fullerene solution is 1.5 ml, the mass concentration of HAuCl4 is 0.01 g / mL, the usage is 70 μL, and the deionized water used is 20 mL.

4. The method for detecting chlorpyrifos in tea leaves according to claim 1, characterized in that: In step (3), the microwave digestion process temperature is 180 DEG C, and the pressure is 1.0 MPa.

5. The method for detecting chlorpyrifos in tea leaves according to claim 1, characterized in that: In the step (4), the chlorpyrifos aptamer solution is 0.14 mmoL / L, the CD Au is 18 μg / L solution, and the mixture is uniformly mixed and left to react for 5 min, so that the aptamer is more completely and better adsorbed to the surface of the carbon dots.

6. The method for detecting chlorpyrifos in tea leaves according to claim 1, characterized in that: In step (6), the concentration of glucose is 45 mmoL / L, the concentration of HCl is 0.59 mmoL / L, the concentration of HAuCl4 is 0.65 g / L, and the solution is mixed and constant volume to 10 mL.

7. The method for detecting chlorpyrifos in tea leaves according to claim 1, characterized in that: In step (7), the water bath heating temperature is 70 DEG C, and the reaction time is 18 min.

8. The method for detecting chlorpyrifos in tea leaves according to any one of claims 1-7, characterized in that: In step (8), take 2 mL of the solution after termination of the reaction in step (7), and the concentration of victoria blue B solution is 0.125 μmol / L.

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